Process for the preparation of a formulation of dihydroartemisinin for the control of wide spectrum of malaria

ABSTRACT

The present invention relates to a synergistic formulation useful for the control of wide spectrum of malarial infections, which comprises a pharmaceutically effective amount of dihydroartemisinin and a vegetable oil.

This appln is a Div. of Ser. No. 09/264,352 filed Mar. 5, 1999, U.S.Pat. No. 6,214,864.

FIELD OF THE INVENTION

This invention relates to the formulation of artemisinin derivative(Dihydroartemisinin) for the emergent treatment and control ofuncomplicated/severe complicated/cerebral and multi-drug resistancemalaria. Dihydroartemisinin is a lactol derivative of artemisinin(Qinghaosu), the principle antimalarial constituent of the plantArtemisia annua.

BACKGROUND OF THE INVENTION

Malaria is caused by blood protozoa of the genus Plasmodium. The fourspecies of Plasmodium that infect humans are Plasmodium vivax, P.malariae, P. ovale and P. falciparum, the last one is responsible forproducing severe complications and cerebral malaria, which can cause thepatient to lapse into a coma and ultimately leads to death. In manyparts of the world, strains of P. falciparum have emerged which areresistant to chloroquine, mefloquine, halofantrinc, quinine andsulfadoxin+pyrimethamine combination, and sulfamethodpyrazine+pyrimethamine combinations the trusted drugs of choicefor control of malaria. Like-wise P. vivax infections resistant tochloroquine are emerging in different countries. More than 270 millionpeople suffer from the disease, and 1.2-1.7 million deaths occur yearly.Mortality is more among children under 5 years of age who are speciallysensitive because of their lack of immunity to the disease (Ziffer H;Highet RJ and Klayman DL; Artemisinin an endoperoxide antimalarial fromA. annua. Progress in the chemistry of organic natural product: Herz W(Ed.). Springer-Wien New York, 1997. P. 121-214).

Severe complicated malaria is a life-threatening condition and comatosecerebral malaria cases need emergency parental therapy. Severe P.falciparum infections are common in both rural and urban areas and themanagement of these cases becomes difficult because of emerging problemsof drug resistant infections. The comatose cases generally do notsurvive more than 72 hrs and therefore, require urgent antimalarial drugtherapy. The suppository preparations of a variety of antimalarials canbe effectively used as emergency treatment in rural areas as well asprimary health care centres and these can be easily administered even byunskilled health workers in remote areas. Several reports have beenpublished which support the observation that rectal suppositorypreparations of artemisinin have powerful effect in reducing thefalciparum parasitaemia in critically sick and severe cases includingcerebral complications (Vinh et al. (1997); Trans Roy Soc. Trop. Med.Hyg. 91, 465-467; Li et al 1985. J Trad. Chinese Med. 5, 159-161).

The endoperoxides are a promising class of antimalarial drugs which maybeet the dual challenges posed by drug parasites and the rapidprogression of severe malarial illness and complications which can provefatal unless emergency treatment is instituted. Artemisinin, is asesquiterpene lactone containing an endoperoxide bridge (—O—O—C) and isunique among the antimalarial drugs. Dihydroartemisinin (DHA) is thereduced lactol derivative of artemisinin and the semisyntheticderivatives (artemether, arteether, artesunate and artelinate) areethers or esters of the lactol. In general, the endoperoxides present inall these derivatives, have several advantages over existingantimalarial drugs. These derivatives show little or no cross-resistanceto existing anitmalarials. The endoperoxides are fast-acting and clearthe periphera blood of parasites more rapidly than other available drugsand finally resistance to the endoperoxides has not yet developed,despite widespread clinical trials. (White N.J., 1994, ArtemisininCurrent Status: Trans R. Soc. Trop Med Hyg. (88 Suppl). 53-54). Theattractive feature of the drugs (arteether, artemether) is the lack ofsystemic neurotoxicity at the clinically prescribed doses (Looaresuwan,S. et al. 1997 Acta. Tropica. 67, 197-205).

Prior art

Dihydroartemisinin (DHA) is the simplest semisynthetic derivative ofArtemisinin, the principle antimalarial constituent of medicinal plantArmeisia annua (Warburton, D. (1984) Handbook of ExperimentalPharmacology, MaCmillan NY pp. 471-495; Hoffman, S. L. (1986). Clin.Trop. Med. Communicable Dis. 1, 171-274). It has considerable activityin vivo and in vitro and is 3.8 to 5 times more potent than artemisinin(de Vries, P. et al (1996). Drugs 52, 818-836; China CooperativeResearch group on Qinghaosu, (1982b) J. Trad. Chin. Med. 2, 17-24); Gu,H. M. et al, (1984) Trans. Roy. Soc. Trop. Med. Hyg, 78, 265-270). Invitro bioassay against drug-resistant P. falciparum cultures (W-2Indo-China and D-6 Sierre Lone strains) showed that direct antimalarialactivity of DHA is superior to that of artemether and arteether β (Lin,A. J. et al (1987) J. Med. Chem. 30, 2147-2150). However, because of itspoor solubility in water, DHA had only been formulated as an oralpreparation (Tablet) (Li, Q. G. et al (1998) J. Pharm. Pharmacol. 50,173-182). DHA has been chiefly used for making semisynthetic derivativessuch as artemether (AM) and arteether (AE) which are soluble in oil, andthe water soluble drug artesunate (AS) (Luo, X. et al, (1984) Helv.Chem. Acta. 67, 1515-1522; Lin, A. J. et al, (1987) J. Med. Chem. 30,2147-2150; Brossi, A. et al, (1988) J. Med. Chem. 31, 645-650). Zhao andSong (1993 Yao Hsuch Pao, 28, 342-346) compared the pharmacokinetics oforal dihydroartemisinin (DHA) and qinghaosu (QHS) Tablets in humanvolunteers and reported very high bioavailability of DHA (oral tablets)as compared to QHS (oral) and serum level after QHS were only 1.6-10.8%that of oral DHA preparation. For oral administration DHA was reportedto be far superior blood schizontocide compared to QHS. Qi-Gui Li et al(1998 J. Pharm. Pharmacol. 50, 173-182) compared the oral bioactivity ofdihydroartemisinin (DHA) with artemether, arteether and artesunic acidin rats and concluded that maximum plasma concentration was produced byoral DHA (769±218) in comparison to artemether (381±113), arteether(324±10) and artesunic acid (208±25 ng ml⁻¹). Further area under plasmaconcentration time curve (ngh ml⁻¹) was highest with oral DHA 9615±56)as compared to artemether (306±52), arteether (298±68) and artesunicacid (217±34). Elimination half life (h) was longer for DHA (4.94±0.73)as compared to artemether (2.04±0.18), arteether (1.79±0.47) andartesunic acid (1.34±26 h). DHA was reported to be active metabolite ofother artemisinin derivatives such as artemether (AM), arteether (AE),Artesunate (AS) Artelinic acid (AL).

Benakis et al (1996) administered (60 mg dose representing half of dailydose) oral dihydroartemisinin tablets to six uncomplicated falciparummalaria cases and the peak plasma levels of dihydroartemisinin(0.26-0.71 μg/ml) were recorded between 1‥2 hrs in four subjects.Thereafter, plasma level dropped and there was complete excretion by 6hrs. Elimination half-life of DHA was 0.71-1.45 h, drug was welltolerated in 2 subjects while 4 subjects experienced mild headache,nausea or vomiting. One subject showed gastrointestinal hemorrhage andstupor 30 min after drug (Tablet) administration. Benakis et al (1996 J.Trop. Med. Hyg. 24, Suppl 1, 7-11) suggested that the development of asustained release form of DHA could overcome the adverse drug reactionof table form of drug.

Benakis et al (1977) also reported that following oral artesunateadministration, the maximum dihydroartemisinin, plasma level producedwas much higher 0.57±0.18 μg/ml compared to that of parent compoundartesunate which recorded peak level of 0.12±0.11 μg/ml. It may beemphasized, that DHA level was 5-fold higher compared to that ofartesunate and that DHA is the main antimalarial principle followingartesunate administration. Further it was pointed out that forartesunate type of antimalarials, dose administration (every 3 hourly)would be necessary because of a very short half-life of the drug.However, Bethell et al (1977 Trans R. Soc. Trop. Med. Hyg. 91, 195-198)had observed wide variation in peak plasma concentration of DHA (664 ngof DHA/ml, 95/Cl 387-9410, range (79-1394) in Vietnamese P. falciparumcases who were administered oral artesunate.

The initial studies with artemisinin suppositories were carried out inChina for the control of P. falciparum. The suppository treatment wasfound satisfactory as shown by fever clearance time (15-39 h) andparasite clearance time (35-52 h) (Li et al, 1989, IV World Cong. Clin.Pharmacol. & Therapeutics, Munnich-Heidel-berge, Germany, July 1989).Arnold et al (1990; Trans Roy. Soc. Trop. Med. Hyg. 85, 499-502)reported that artemisinin suppositories (Containing 2800 mg total drugadministered in 56 h) produced complete parasite clearance in 41.8 h incases with acute P. falciparum infection, while those receiving quinine(Oral) at 1500 mg dose daily×14 days would take 68.1 for parasiteclearance.

Hien et al (1991 Trans. Roy. Soc. Trop. Med. Hyg. 84, 499-502) hadreported that artemisinin (600-2200 mg) suppositories rapidly clearedasexual P. Falciparum parasites in children and confirmed the problem ofhigh incidence recrudescence with this drug. Suppositories could be usedas presumptive treatment to prevent the development of highparasitaemia. In the study 10 children receiving 10-mg/kg artemisinintook 18.9±4.7 hrs to achieve initial 95% clearance of parasitaemia.

Astesunate suppositories (1600 mg dose administered over 3 days) wereevaluated by Looareesuwan et al (1997, Am J. Trop. Med. Hyg. 57, 348-53)and (200 mg×3 days) by Kyaw et al. 1996. According to these workers, theartesunate suppositoreis achieve complete parasite clearance at afantastic speed (16-36 h) as good as that achieved by oral tablet ofartesunate. Patients with cerebral symptoms and altered conscious levelsgenerally give a good response and recover. Rectal suppositories wereable to control severe complications of P. falcipurum cases better thanim artemether as shown by time to regain full consciousness in comatosecases.

Artemisinin suppository treated cases look 24 (18-30) h for recoveryfrom coma, while artemether took 47 h (31-63 h). The efficacy of rectalartemisinin suppository was equal to iv artesunate which is consideredto be most fast-acting treatment for comatose cases as reported by Heinet al. (1992). The development of rectal formulation would provide abetter alternative to the oral treatment in terms of quick absorption,high bio-availability and effective plasma level adequate to exertantimalarial effect in the blood.

The fast excretion rate would justify the repeated rectal administrationevery 4 hrs initially to control severe complicated infections, withoutcausing any toxicity. Rectal suppository are simple to administer, easyto store at room temp and its administration does not require anyspecial equipment, and can be given as emergency drug at rural healthcentres throughout the developing world where malaria related mortalityis high.

The water soluble derivative of DHA, namely the artesunate acid has beenfound to be very effective against malaria in vitro and has got lowtoxicity in vivo and in vitro (Yang, Q. et al (1982) J. Trad. Chin.Med., 3, 99-103; Lin, A. J. et al (1987) J. Med. Chem. 30, 2147-2150).Although Artesunate (AS) is 3.2 time more potent than artemisinin and isless toxic than artemether, it has limited stability in solution (Zhou,Z. M. et al (1987) J. Chromato 414, 77-90; Panisko, M. D. and Keystone,J. S. (1990) Drugs, 39, 160-189) and dose must be prepared immediatelybefore administration. Pharmacokinetic data obtained in man, rabbit, ratand dog suggested that artesunic acid is distributed and hydrolysed toDHA by plasma esterase with an elimination half-life of 2-4 min inrabbits and 27 min in dogs (Zhao, K. C. et al (1986) Acta. Pharm. Sin.21, 736-739). Due to its rapid and extensive conversion to DHA,artesunic acid could be considered a prodrug of DHA (Titulaer, H. A. C.et al, Int. J. Pharm. 69, 83-90,1991). The pharmacodynamic activity ofartesunate is due to the metabolite (DHA) rather than to be administeredproduct.

Karbwang, J. et al (1998) (Ann. Trop. Med. and Parasitol, 92, 31-36)studied the pharmacokinectics of artemether (oral) and its conversion toDHA in P. falciparum cases and concluded that DHA was believed to be themain determinant of the successful treatment, suggesting thatanitmalarial activity corresponded to DHA level in plasma. Plasma levelof DHA metabolite was nearly three fold higher in patients withsensitive P. falicparum infection. The mean ratio of inhibitoryactivities of artemether vs. DHA against P. falciparum isolates inThailand was 1:2.9 within 6-12 hrs, the plasma artemether levelsdecreased fast, while DHA level was still higher. However, bothartmether and artesunate are susceptible to breakdown by humidity, lightand acidic conditions at room temperature. An aqueous solution of sodiumartesunate at pH 7-8 hydrolyses within 1 h to DHA.

DHA is a major metabolite of artemether, arteether and artesunicacid/artesunate in vivo (de Vriens, P. J. and Dien, T. K. (1996) Drugs,52, 181-836; Chi, H. T. et al (1991) Biol Mass Spectrum 20, 609-628;Zhou, Z. M. et al (1987) J. Chromatog, 414, 77-90; Li, Q. G. (1998) J.Pharm. Pharmacol. 50, 173-182). The high rate of malaria recrudescenceassociated with other artemisinin derivatives has been ascribed at leastpartly to short plasma half-life.

The pharmacodynamic activity of artemether in healthy adults wasreported to be due to the metabolite DHA rather than administeredprodrug. The main metabolite DHA is approximately three times as activeas the parent compound in terms of antimalarial activity against P.falicparum (Teja-Isavadharm, P. et al (1996) Br. J. Clin. Pharmacol. 42,599-604). The contribution of other unidentified metabolites toantimalarial activity was reported to be negligible ((Lee, I. S. andHeefford, C. D. (1990) Pharmacol Ther.48, 345).

In vivo conversion of arteether (AE), artemether (AM) and artesunate(AS) to DHA in rats was compared via all the three routes (oral, im andrectal) of administration, and the conversion rate was faster with ASfollowed by AE and AM. The high conversion of AS to DHA can be explainedby the fact that AS and DHA show same in vitro antimalarial potencyagainst chloroquine sensitive and chloroquine resistant strains of P.bergei. Although the DHA level obtained after dosing with AM, AE and ASwere less than 2.6% those after im dosing with DHA, the highantimalarial activity of DHA probably contributes significantly tooverall antimalarial activity of these drugs in vivo (Li, Q. G. et al(1998) J. Pharm. Pharmacol, 50,173-182).

The bio-availablility of artemether in healthy subjects given drugs byintramuscular and intra-rectal routes showed that plasma profile of itsactive metabolite dihydroartemisinin following intarcctal (ir)administration suggested that this route should be assessed as analternative to intramuscular route in the rural tropics (Teja Isavadharmet al. 1996. Br. J. Clin. Pharmacol. 42, 599-604). The conversion ofartesunate to its active metabolite dihydroartemisinin appears unique inantimalarial pharmacology because of its rapid clearance rate. (Benthellet al. b1998, Br. J. Clin Pharmacol. 45, 123-129).

The im bio-availability of AM and AE were very low, indicatingincomplete absorption of these drugs atleast during first 8 h after imadministration. The bio-availability of DHA on the other hand, is midwaybetween other oil soluble drugs and water soluble drugs. The relativelyslow and incomplete bio-availablity of both 1M and oral preparation ofartemether is of some concern as this is a front line treatment forsevere and complicated falciparum malaria. The im absorption ofartemether in children with severe malaria (Cerebral malaria (CM)accompanied with respiratory distress was reported to be erratic andfive CM cases did not show plasma DHA levels in one study (Murphy, S. A.et al.(1997) Trans. Roy. Soc. Trop. Med. Hyg. 91, 332-334). In view ofthe impaired absorption of artemether in children, there is an urgentneed to develop an effective antimalaria for children. Oral artemetherhas been reported to undergo inadequate drug absorption in P. falciparumcases which resulted in recrudescence (Bangchang, K. N. A. et al (1994)J. Chem. Pharmac. 37, 249-253). Clinical investigators therefore,advocate the replacement of artemether with iv artesunate preparationfor treatment of severe malaria in children (Murphy, S. A. et al (1997)Trans. Roy Soc. Trop.Med. Hyg. 91, 332-334).

The plasma level of DHA by im route were sustained over 30 hr andparasitidal effect was maintained for much longer time period comparedto oral or Intarectal route (DHA activity by im route, AUC 3445 n Mlol⁻¹ h compared to oral route 3855 n mol⁻¹ h but activity due to DHA wasprolonged beyond 30 h while the oral dose provided effective level upto10 h (Teja-Isavadharm, P. et al (1996) Br. J. Clin. Pharmacol. 42,599-604).

The earlier work on artemisinin, arteether, artelinate, artesunateadministered by im formulations and arteether and DHA administered byoral route have shown good gemetocidal activity both in animal model andagainst P. falciparum. These drugs have potential for the interruptionmalaria transmission. (Dutta, G. P. et al (1989) Chemotherapy (Bansal)35, 2000-2007; Tripathi, R. et al (1990), Amer. J. Trop. Med. Hyg. 43,571-575; ibid, (1985) 54, 652-654).

Cost is a critical factor in determining use of antimalarial drugs.Artemisiinin derived drugs are now available commercially in a fewcountries but they are restricted because of high cost, eg., oralartesunate currently cost about $5-6 per treatment as compared to $1.85for mefloquine and 7° C. for drugs such as chloroquine. Injectableartesunate, artemether and arteether cost even more compared to quinineinjections at less than $2.0 per treatment.

There is undoubtedly a real need for an effective new antimalarial drugbased on artemisinin or its semi-synthetic derivatives with improvedabsorption, bio-availability, high plasma drug concentration for longerduration of time and cost effectivenss for the treatment of multi-drugresistant and severe complicated/cerebral malaria.

The main object of the present invention is to develop a formulation ofdihydroartemisinin for the control of wide spectrum of malaria.

Anotehr object of invention is to develop improved safe formulation,less expensive, for uncomplicated malaria infections as well as for thecontrol of multi-drug resistant malaria and emergent treatment of severecomplicated cerebral malarial infections.

Still another object of invention is to develop a formulation which willbe able to stop malaria related mortality among children and adults.

SUMMARY OF THE INVENTION

The present invention is to develop an improved formulation ofartemisinin derivative for the control of both uncomplicated malaria andfor the emergent treatment of severe complicated and cerebral malariacases and for the treatment of multi-drug resistant malarias. Thisformulation comprises preparation of artemisinin derivative in sterile,neutralized refined oil which would exert fast acting bloodschizontocidal activites in adults and children and has long shelf-life.The antimalarial profile of the formulation given by different routes(rectal, intramuscular and oral) has confirmed high activity.

DETAILED DESCRIPTION OF INVENTION

For the present invention we have chosen to develop a formulation ofdihydroartemisinin for the following reasons: The product is superior tothe original molecule, artemisinin in terms of efficacy.Dihydroartemisinin is simplest one step, high yield and economicsemi-synthetic derivative of artemisinin. Dihydroartemisinin is known tobe an active metabolite of artemether, arteether, artesunic acid, andartelinic acid which is responsible for the antimalarial activity ofthese drugs (Lee, Q. G. et al (91998) J. Pharm. Pharmacol. 50, 173-182).

The antimalarial profile of the compound given by oral route has beenconfirmed by high activity of dihydroartemisinin formulation. The oralroute of administration is important for treatment in villages as wellas in rural and urban areas. The oral formulation gives 100% cure rateagainst MDR P. yoelii nigeriensis malaria and the efficiency is betterthan QHS (artimisinin) and its other derivatives used by earlier workerswhich are effective upto 50-90% at exceptionally high doses. Comparableantimalarial effects can be achieved with DHA at safe and low dosewhereas high doses of QHS upto 5.0 g dose gives cure rate 55%,Artemether upto 700 mg give cure rate 90% and artesunate upto 750 mggives cure rate 70-90%. Safety of DHA formulaation is better thanartemether and artesunate.

The route of administration is important for the emergency treatment ofsevere complicated/cerebral malaria (Shen et al 1989, Antimalarial drugdevelopment in China 31-45; Hein, T & White NJ (1993) Lancet 34,603-608). The DHA rectal formulation is simple to administer, easy tostore and its administration does not require any special equipment. Inorder to achieve higher efficacy, the repeated rectal administration ofthe drug every 4-6 hrs initally, to control severe complicatedinfections without caussing any toxicity.

An intramuscular DHA formulation would be effective in controllingsevere complicated P. falciparum infections. The im formulation wouldnot need an intravenous infusion apparatus. Intramuscular DHA is readyto use, unlike intravenous artesunate which is available in the form ofan ampoule of lophilizate and an ampoule of solvent which must be mixedbefore use. The prepared solution of artesunate is not very stable onstorage and injection must be given rapidly, Moreover, the risk ofaccidental overdose is certianly less in im than with an iv prepartion.

According to Li et al (1988, J. Pharm. Pharmacol. 50, 173-182), themaximum plasma concentration by im route attained in rats with DHA isvery high in comparision to arteether as well as artemether (DHA1579±443; AM 692±234 and AE 160±12.4). From the Pharmacokinetic point ofview the half-life of DHA is longer than that of other artemisininderivatives (DHA 4.44±0.27 h vs AE 362±0.9 h vs AM 1.78±0.80 h). Whenthe cytotoxicity of artemisinin and dihydroartemisinin were comparedwith ethers and esters of dihydroartemisinin, it could be seen thatdihydroartemisinin was significantly less cytotoxic than artemether,arteether, artelinic acid and sodium artelinate (Woerdenbag et al. J.Nat. Prod. 56, 848-56, (1993). The absorption (%) of DHA within 8 h is85.4±12.1 which is much higher than AM (54.2±25.9) and AE (34.0±9.6).

Comparison of Main Pharmacokinetic parameters of DHA, AM, AE and AS inrates after single IM dose

DHA Di- hydro- AM arte- Arte- AE AS Parameter misinin mether ArteetherArtesunate 1) Maximum 1579 ± 692 ± 160.7 ± 12.4  1650 ± 446  plasma 443234 Concentration (ng ML⁻¹) 2) Time of 17.5 ± 28.8 ± 41.4 ± 14.7 15.0 ±4.1  maximum 2.89 11.8 plasma Concentration (min) 3) Area under 2719 ±1007 ± 285.7 ± 80.5  773 ± 398 plasma 38.5 48.1 concentration (h ml⁻¹)4) Distribution 0.73 ± 0.23 ± 0.34 ± 0.07 0.15 ± 0.06 half life (h) 0.130.04 5) Elimination 4.44 ± 1.78 ± 3.62 ± 0.09 0.54 ± 0.14 half life (h)0.27 0.80 6) Mean 0.21 ± 0.14 ± 0.25 ± 0.06 0.11 ± 0.07 absorption 0.040.04 time (h) 7) Absorption 85.4 ± 54.3 ± 34.0 ± 9.6  104.7 ±     0-8 h(%) 12.1 25.9

In contrast to above artemisinin derivatives dihydroartemisinin (DHA)seems to be a good antimalarial candidate as it has the highest plasmaconcentration, the highest binding capacity in RBC, the longestelimination half life and the lowest toxicity. The absorption of DHA in8 h is very high in comparison to other drugs. DHA is a major metaboliteof all these drugs and antimalarial activity of these drugs is due tomajor metabolites DHA. DHA showed the low rate of recrudescence incomparision of artemether and arteether. Therefore, thedihydroartemisinin has been synthesized and stable sormulation withenhance bio-availability was prepared for the control and emergenttreatment of uncomplicated/severe complicated/cerebral malaria andmulti-drug resistant malaria infections.

Accordingly, the present inventoin provides a formulation of thedihydroartemisinin for the control of wide spectrum of malaria whichcomprises (a) preparation of dihydroartemisinin from artemisinin byknown method, (b) dissolving dihydroartemisisin in sterilzed neutralrefined vegetable oil by heating at 70-90° C. for 2-4 min and coolingthe solution at room temperature to obtain the desired formulation.

The invention further provides a formulation which shows no adversegastric effects, hemorrhage etc. which are found in the treatment withdihydroartemisinin tablet.

The invention further provides a formulation which provides maximumbio-availability in comparison to other artemisinin derivatives,selected from artemether and arteether.

The invention further provides a formulation which has more than 80%absorption of compound within 8 h, which is much higher than otherartemisinin derivatives (artemether, arteether).

In another embodiment of the invention, wherein dihydroartemisinin andthe vegetable oil is present in a ratio of 0.22-0.33:1 w/w.

In another embodimetn of the invention, the refined vegetable oil usedfor prepartion, is selected from groundnut oil, sesame oil, tea oil etc.

In another embodiment of the invention, the dihydroartemisinin used inthe preparation appears in mixture of α and β tautomers with the ratioof and β in the solvent chloroform (1:1) and methanol (2:1).

In another embodiment of the invention, the dihydroaretmisinin used inthe formulation has the following characteristics: MP:(153-154° C.), MW:284, MF: C₁₅H₂₄O₅.

The invention provides a formulation which is viscous in nature andlight yellow in colour.

The invention further provides a formulation which is safe, welltolerated and better than artemether and artesunate of its safety.

The invention further provides a formulation which has shelf life ofatleast two years;

The invention further provides a formulation which as 200-300 times moreaccumlation in the malarial infected red blood cells than the normal redblood cells showing the high antimalarial potential of this formulation.

The invention further provides a formulation which is fast acting,having blood schizontocidal activity and useful for the treatment ofuncomplicated/severe complicated/cerebral and multi-drug resistantmalarial infections.

The invention further provides a formulation which is effective againstmulti-drug resistant Plasmodium yoelii nigerienses parasite which isresistant to high oral dose of chloroquine, amidoquine, mapaerine,mefloquine, quinine and halofantrine.

The invention further provides a formulation which is the safe subsituteof Primaquine and useful for interrupting transmission of P. falciparumand other malarial infections.

The invention further provides a formulation w hich can be administeredthrough oral, intrarectal and intramuscular routes and reduce thegamatocidal effect.

The invention provides a formulation from which has no adverse mathemoglobin type of toxicity which is known to be assocated withadministration of primaquine.

The invention further provides a formulation which is effective againstblood asexual stage as well as sexual stage of the both P. falciparumand P. vivex parasites.

The invention futher provides a formulation which can be used throughoral route for control of uncomplicated Plasmodium falciparum and P.vivex infections.

The invention further provides a formulation which can be used throughoral route for control of chloroquine resistant and halofantrineresistant P. vives malaria.

The invention further provides a formulation which can be used throughoral route for control of multi-drug resistant P. falciparum infectionscaused by chloroquine, mefloquine, quinine, amidoquine and halofentrine.

The invention further provides a formulation which can be used throughoral route for control of treatment of P. malariae and P. ovaleinfections.

The invention further provides a method of treatment of malaria throughoral route by administration of a daily dose in the three divided doses(every 8 hrs) to reduce the recrudescence rate of malaria.

The invention futher provides a formulation which can be used throughintrarectal route for emergent treatment and control of uncomplicated,severe complicated/cerebral and multi-drug resistant malarialinfections.

The invention further provides a formulation which can be used thorughintrarectal route for rectal suppository for the fast action.

The invention further provides a formulation which can be used throughintrarectal route for the emergent treatment of severe, complicated andcomatose cerebral malaria infection to prevent progression of thedisease.

The invention further provides a formulation which can be used throughintrarectal route for the treatment of children in severe complicatedmalaria and prevent mortality and the progression of severity of thesedisease.

The invention further provides a formulation which can be used throughintrarectal route for as emergent and life saving treatment of severecomatose and cerebral malaria cases.

The invention further provides a method of treatment of malaria whichcan be used through intrarectal route by administration of theformulation daily in two or three divided doses at 8-12 h interval tocontrol the malarial infection.

The invention further provides a formulation which can be used throughintramuscular route for emergency life saving treatment for severecomplicated and cerebral malaria.

The invention further provides a formulation which can be used throughintramuscular route for the fast recovery of the comatose patients.

The invention futher provides a formulation which can be used throughintramuscular route for the complete cure of the uncomplicated, severecomplicated/cerebral malaria and multi-drug resistant malariainfections.

The invention further provides a formulation which can be used throughintramuscular route to prevent and interrupt malarial transmisson tostop multi-drug resistance in the patients.

The invention further provides a method of treatment of malaria throughintramascular route by administration of the formulation by dailyinjection in two divided doses for five consecutive days for adults.Doses will be correspondingly reduced for children and infants.

This invention is described in detail in the following example. However,it should not be construed to limit the scope of the present inveniton.

EXAMPLE Chemistry, formulation and Antimalarial evaluation ofdihydroartemisinin (DHA):

a) Preparation of dihydroartemisinin from artemisninin:

Artemisinin (10 g) in 250 ml of methanol, was cooled in an ice bath to0° C. To the stirred solution was added 4 g of NaBH₄ over a period of 30min. The reaction mixture was stirred for 1 h after complete addition ofNaBH₄. The crushed ice was added to the reaction mixture which produceda precipitate immediately. The white precipitate was filtered and washedwith cold water. The precipitate was dissolved in CH₂Cl₂ (250 ml) anddried over anhydrous sodium sulphate. The solvent was evaporated undervacuum and residue was recrystallized with ethylaceteate-hexane (1:3)solvent which yielded (9.1 gm) of pure crystalline substance of mp151-153° C. The dihydroartemisinin MF: C₁₅H₂₄O₅; MW 284) has been foundto be a hemiacetal and exist as a mixture of a α and β anomers whoseratio is solvent dependent. When dihydroartemisinin is crystalline, thehydroxyl at β-position but when in solution, a mixture of epimers isformed. The ratio of α and β isomers in the ¹³CNMR of dihydroartemisininwas found to be (1:1) in CDCl₃ and (2:1) in CD₃OD solvent. (Pathak etal. Ind. J. Chem. 34B, 992-93 (1995). The material was characterized byspectral methods (IR, ¹H NMR, ¹³C NMR & mass) and authentic sample.

b) Formulation of dihydroartemisinin (DHA):

Dihydroartemisinin (DHA) 200 mg was taken in sterile refined neutralized(10 ml) groundnut oil and dissolving by mild heating at 80-90° C. andcooling the solution at room temperature. The formulation was stored atroom temperature till use. It has been found to be stable at roomtemperature for several months.

(c) Antimalarial efficacy:

Test Parasite: Multi-drug resistant Plasmodium yoeli nigerienes (Rodentmalaria strian). The P. yoeli nigerienses is a multi-drug resistantstrain which is resistant to high oral doses of chloroquine (128mg/kg×4), amodiaquine (128 mg/kg×4), meperaine (128 mg/kg×4), mefloquine(128 mg/kg×4), quinine (400 mg/kg×4) and halofantrine (32 mg/kg×4). Thisparasite produces 100% mortality in 20-15 g Swiss mice and it is highlyvirulent for mice. It is ideal parasite for antimalarial evaluation asit produces acute infection with a spectrum of wide range of multi-drugresistance similar to the spectrum of resistance reported in the fieldisolates of P. falciparum.

(i) Antimalarial (blood Schizontocidal) activity of dihydro-artemisininformulation by rectal route:

DHA formulation was evaluated in vivo in Swiss mice (20 g). The micewere infected intraperitoneally with parasite (multi-resistant strain ofP. yoelii nigeriensis) and the infected mice were treated for 5-7consecutive days (Day 0 onwards) with each mouse does (0.2-0.3 ml oil byrectal route) of a DHA formulation prepared in neutralized ground nutoil. For higher doses (15 and 20 mg/kg) the drug was given in divideddoses by rectal route. The tail blood smears of the mice were examinedperiodically after Giemsa staining upto 30 days post-infections. DHAformulation given by rectal route has shown curative effect againstmulti-drug resistant rodent infection and severe/virulent/ethal malariainfection as shown by complete absence of parasitaemia till 30 ofobservation (Table 1).

TABLE 1 Blood schizontocidal activity of dihydroartemisinin againstmulti-drug resistant P. yoalii nigeriensis infection in Swiss mice (20 ±1 g) by rectal route. Dose mg/kg Parasitaemia % Cure-rate Treatment ×days Day 5 Day 7 Day 30 (%) DHA  5 mg/kg × 7 0.83 ± 1.31 1.12 ± 1.55 25(in oil) (8) 4-ve (8) 4-ve (Rectal route) Doses: 20 mg/kg × 7 0.0 0.0 875-20 mg (8) (8) Control —  6 ± 2.92 68.87 ± 14.57 (8) (8) Note= No. ofmice are given in parenthesis.

Drug is dissolved in sterile ground nut oil for adminisitration.

(ii) Antimalarial (Blood Schizontocidal) activity of dihydro-artemisininformulation by intramuscular route:

DHA formulation was evaluated in vivo in Swiss mice (20±1 g) as well asbaby mice (12±1 g). The mice were infected intraperitoneally withparasite (multi-resistant strain of P. yoelii nigeriensis) and theinfected mice were treated intra-muscrularly for 5 consecutive days (Day0,+1,+2,+3,+4) with each mouse dose (0.2 ml oil injection) of a DHAformulation. The tail blood smears of the mice were examinedperiodically after Giemsa staining upto 30 days post-infection. DHAformulation has shown curative effect against multi-drug resistantrodent infection as shown by complete absence of parasitaemia till 30days of observation (Table 2).

The DHA formulation given by im route is able to completely controlmulti-drug resistant P. yoelii nigeriensis malaria which is highly fatafor Swiss mice. The drug DHA is also effective in controllingsevere/virulent/lethal malaria infection in 20-15 g adult mice as wellas in baby mice (912±1 g).

TABLE 2 Blood schizontocidal activity of dihydroartemisinin, againstmulti-drug resistant strain of P. yoeli nigeriencsis in Swiss mice (20 ±1 g) and in baby mice 12 ± 1 g by intramuscular route Dose Cure rateTreatment Host (mg/kg × days) (%) Expt. I DHA (20 ± 1 g)  5 mg/kg × 5100% (8/8) (IM in oil) Dose 5-30 mg/kg Expt. II DHA (im in oil) (20 ± 1g)  5 mg/kg × 5 100% (8/8) Dose 5-10 mg/kg 10 mg/kg × 5 100% (8/8) Expt.III DHA(im in oil) (12 ± 1 g   5 mg/kg × 5 100% (9/9) Dose 5 mg/kg babymice) Control (20 ± 1 g) Vehicle (oil) Nil (0/20) Safety in 20 ± 1 gmice 30 mg/kg DHA × 5 Safe 8/8 Healthy mice

Drug treatment was started on day 0 (ie day of infection). Mice showingnegative blood slide upto day 30 were considered as cured.

(iii) Antimalarial (blood schizontocidal) activity of dihydroartemisininformulation by oral route:

DHA formulation was evaluated in vivo in Swiss mice (20-25 g). The micewere infected intraperitoneally with parasite (multi-drug resistantstrain of P. yoelii nigeriensis and the infected mice were treated for 5consecutive days (day 0 onwards) with each mouse dose of 0.25 ml (inoil) by oral feeding. The DHA formulation is prepared in neutralizedsterile ground nut oil. The tail blood smears of treated mice wereexamined periodically after Giemsa for recording % parasitaemia. DHAformulation given by oral route has shown curative effective againstmulti-resistant plasmodium which is resistant to high doses ofchloroquine, melfoquine, quinine, amodiaquine, mepacrine andhalofantrine etc. The DHA oral formulation at 30 mg/kg dose×5 days hasshown curative efficacy of 100% against MDR strain of P. yoeliinigeriensis (Table 3).

TABLE 3 Blood schizontocidal activity of dihydroartemisinin againstmulti-drug resistant P. yoalii nigeriensis infection in Swiss mice(20-25 ± 1 g) by oral route. Dose Parasitamia Cure Treatment mg/kg Wt.of (%) (Mean ± SD) rate (%) route × days mice Days 6 7 9 30 DHA (oral 30mg/kg 25 ± 1 g — — — 100 in oil) × 5 (12) (12) (12) Control 1 Vehicle 20± 1 g — 68.87 ± 75 Nil oil 14.57 (1) (8) 2 Vehicle 25 ± 1 g 10.12 ± —87.5 ± Nil oil 3.09 3.53 (8) (2) No. of mice are given in parenthesis.

The improved formulation of dihydroartemisinin-the subject matter ofthis patent offers a number of advantages.

1. The product dihydroartemisinin as oil formulation will be a fastacting, blood schizontocide which would be able to controluncomplicated/severe complicated/cerebral and multi-drug resistantmalaria infections.

2. The formulation given by im and rectal route can be used in ruralareas where facilities such as constant drip generally used for ivadministration of comparable molecule iv artesunate are not available.

3. The rectal administration can be repeated given through syringe orcatheter by the family member in village or by paramedical personals aspresumptive treatment to control the severity of malaria infections andas emergency treatment to comatose malaria cases and prevent deaths fromcerebral involvement in complicated cases.

4. The administration of the formulation by different routes willincrease the plasma drug concentration for effective antimalarialtreatment. Because of the high efficacy of the formulation IMadministration can be given to control the complicated casescerebral/severe/multi-drug resistant cases.

5. The DHA formulation given by rectal followed by IM route or oral,will exert higher level of antimalarial activity.

6. P. vivex sensitive as well as resistant to chloroquine and P.malariae/P. ovale infections, can be also controlled by using thisformulation and blood stage parasitaemias effectively controlled inuncomplicated cases by oral formulation because of its high efficacyagainst MDR infection.

7. The oil based oral formulation of DHA would not produce any gastricadverse reactions as reported for DHA Tablets.

8. This formulation will be economically cheaper than the comparableparental preparations like artemether, and artesunate (iv).

9. Safety of this compound on the basis of LD₅₀ is comparable toarteether and better than artemether, and artesunate.

10. The formulation is stable at room temperature for longer period.

11. The DHA formulations will be able to prevent deaths of both childrenand adults due to malaria complications and stop the progression ofcomatose condition because of their higher antimalarial efficacy,bio-availability and plasma conc.

What is claimed is:
 1. A process for the preparation of a formulationuseful for the control of wide spectrum of malaria comprising (a)preparation of dihydroartemisinin from artemisinin by known method, (b)dissolving dihydroartemisinin in sterilized neutral refined vegetableoil by heating at 70-90° C. for 2-4 min. and (c) cooling the solution atroom temperature to obtain the desired formulation.
 2. A process for thepreparation of a formulation as claimed in claim 1, wherein thevegetable oil used is selected from groundnut oil, sesame oil and teaoil.
 3. A process as claim in claim 1, wherein the dihydroartemisininused comprises a mixture of α and β tautomers in a solvent.
 4. A processas claimed in claim 1, wherein the ratio of α and β tautomers ofdihydroartemisinin to the solvent may be 1:1 or 2:1.
 5. A process asclaimed in claim 1, wherein the solvent used may be selected fromchloroform and methanol.
 6. A process as claimed in claim 1, whereindihydroartemisinin and the vegetable oil is present in a ratio between0.022-0.033:1 w/w.